Protein 4.1 is an 80 kilodalton (kd) phosphoprotein originally identified as a major element in the cortical cytoskeleton of erythrocytes. Protein 4.1 is thought to add strength and flexibility to erythrocyte membranes by linking the spectrin-actin cytoskeletal scaffold to the cytoplasmic domains of transmembrane proteins, such as the glycophorins. Inherited abnormalities of protein 4.1 are associated with congenital hemolytic anemias, demonstrating the importance of this protein to erythrocyte integrity. Recent immunochemical evidence has suggested that multiple related forms (isoforms) of protein 4.1 are detectable as immunologically cross-reacting forms in several tissues. During the past 2 years, we have demonstrated that protein 4.1 consists of a complex family of distinct but related isoforms exhibiting differential expression in several different cell types. By utilizing comparative cDNA cloning, molecular hybridization techniques, and antipeptide antibodies, we have shown that these isoforms differ by insertion or deletion of at least 9 "cassettes" (Motifs) 17-129 nucleotides long into the mature mRNA. Isoform diversity appears to arise by alternative mRNA splicing. We have identified a erythroid-specific splicing event that inserts amino acids important for binding of protein 4.1 to erythroid spectrin-actin, and 2 mRNA splicing events in the 5' untranslated region of the mRNA that allow for synthesis of a "135 kd" large isoform. We have also initiated studies in the region of the glycophorin binding domain. We now propose further studies of structure/function relationships among different protein 4.1 isoforms; we also wish to continue our attempts to define the tissue distribution and metabolism of different isoforms. Regions of the molecule provisionally identified as spectrin-actin, fodrin-actin, and glycophorin binding sites will be altered by site directed mutagenesis, and binding functions assayed in cell free systems and surrogate host cells. The importance of phosphorylation as a means for enhancing reversibility of binding will be assessed by altering phosphorylation sites. Studies of the tissue distribution and intracellular localization of the large protein 4.1 forms will be continued. Metabolic labeling studies will be performed to determine effects, if any, of inclusion or loss of specific motifs on translation rate, stability, and intracellular trafficking of isoforms or reporter proteins attached to the novel amino terminus of the 135 kd form. The regulation of protein 4.1 production during the cell cycle and growth arrest will be assessed, as will the tissue distribution of specific isoforms by in situ hybridization. These studies should improve our understanding of the biological importance of multiple protein 4.1 isoform production, and the range of functions performed by protein 4.1 within specific cells and tissues.